(I) Disease Models and Pathogenesis Goal: To develop disease/infection models for studying pathogenesis and research on vaccines/therapeutics. Rodents as disease models: The Syrian hamster has been broadly used in infectious disease research but research tools are limited. We have developed quantitative real time RT-PCR for monitoring hamster immune response genes (Zivcec 2011) and sequenced the transcriptome of the Syrian hamster. Annotation is still ongoing in collaboration with RTB (Dr. S. Porcella). The Andes virus hamster model is characterized by a strong suppression of innate immune responses during the early stage of infection and massive activation of pro-inflammatory and Th1/Th2 responses during the symptomatic phase suggesting that infection-derived immune modulation is important to pathogenesis (Safronetz, in revision). The lethal Syrian hamster model for Nipah and Hendra virus infections is characterized by acute severe respiratory distress and severe neurological symptoms. Respiratory symptoms were more prevalent in animals challenged with a high dose, whereas animals challenged with a low dose mainly showed neurological signs of infection (Rockx 2011). We established an acute disease model for Crimean-Congo hemorrhagic fever (CCHF) using mice lacking the type I interferon receptor. This model recapitulates most hallmarks of human CCHF disease including coagulation abnormalities (Zivcec, in preparation). The emergence of Reston ebolavirus (REBOV) in domestic swine in the Philippines has caused a renewed interest in its pathogenicity. We have established a mouse disease models based on STAT1-/-mice, which may allow future pathogenesis studies (deWit 2011). Nonhuman primates as disease models: We have established lethal disease models in African green monkeys for Nipah and Hendra virus infection. Animals showed severe lesions in lungs and brains which are the main target organs of the infection. The primary cause of death is severe respiratory distress (Rockx 2011). The virulence of a new Lassa strain from Mali was assessed in cynomolgus macaques in comparison with well studied isolates from Sierra Leona and Liberia. This virus (Soromba-R) was found to be less pathogenic which might be explained by a stronger early innate immune response (Safronetz, in preparation). (II) Vaccines and Therapeutics Goal: To develop and characterize fast-acting vaccines and targets for therapeutic intervention for emerging/re-emerging viruses. Our main platform is based on attenuated recombinant vesicular stomatitis virus (rVSV) vaccine vectors. For post-exposure treatment of filovirus infections with rVSV vectors we could increase the treatment window to 24 and 48 hours post-infection with 80 and 30% success, respectively. This supports the use of the rVSV in cases of emergencies such as laboratory exposures (Geisbert 2010). Cross-protective vaccines would be very desirable for filoviruses in Central Africa due to overlapping endemicity zones. Recent studies in guinea pigs demonstrated that an rVSV vaccine expressing a single filovirus glycoprotein cannot achieve cross-protection, but the use of a bivalent vector expressing a second filovirus immunogen in a two-dose regime strongly increased cross-protective immunity (Marzi 2011). Multivalent vaccine vectors targeting different viruses with overlapping endemicity zones are of even greater public health interest. We performed a proof-of-concept study using a bivalent rVSV vector expressing glycoproteins of Ebola and Andes viruses. Complete protection was achieved against lethal challenge with both viruses in the common Syrian hamster model (Tsuda 2011). We have generated a new rVSV vector expressing Andes virus glycoproteins that provided complete protection in the Syrian hamster model. High titered neutralizing antibodies following a single vaccination seem to be important for protection in pre-exposure vaccination. The vaccine was protective when given three days prior to challenge and remained 90% effective even when administered 24 hours post-challenge. Here, a strong innate immune response seems to be the mechanism of protection (Brown 2011). Ebola is a serious problem for the endangered great ape population in Central Africa. To address vaccine needs for remote wildlife populations we evaluated in a proof-of-concept study the use of a disseminating cytomegalovirus vaccine vector expressing a single Ebola nucleoprotein T cell epitope. The strategy provided complete protection against lethal challenge in the Ebola mouse model. Future studies will focus on a disseminating vaccine vector for nonhuman primates (Tsuda 2011). The cellular cysteine proteases cathepsin B &L are proposed to play an important role in Ebola virus replication. We tested Ebola virus replication in cathepsin B &L knockout mice and could demonstrate that cathepsin cleavage is not necessary for Ebola virus replication. We conclude that other proteases can substitute for cathepsins making it difficult to use proteases as antiviral targets (Marzi, in preparation). We identified a new soluble Ebola virus glycoprotein (ssGP) generated through RNA editing of the glycoprotein gene. ssGP is a disulfide-linked dimer and exclusively N-glycosylated, but a function could not yet be assigned to this new protein (Mehedi 2011). We are currently studying the mechanism of editing and its importance for pathogenesis. Preliminary studies have identified Ebola virus VP30 as a necessary factor for RNA editing. We confirmed that the glycoproteins of pathogenic New World hantaviruses appear to be the primary antagonist of RIG-I directed IFN production. In addition, we showed that the ANDV nucleocapsid protein (NP) serves as the primary antagonist in JAK-STAT signaling (Levine 2010). We tested the in vivo efficacy of a monoclonal antibody (mAb) directed against the G glycoprotein of henipaviruses. This mAb has potent in vitro neutralizing activity against Nipah and Hendra viruses. Intravenous application of the antibody as late as three days post infection completely protected African green monkeys against lethal Nipah or Hendra virus challenge (Bossart 2011). We tested the in vivo efficacy of ribavirin against Hendra virus infection in African green monkeys. Treatment did prolong and alter disease progression but was largely unsuccessful (Rockx 2011). We also tested the effect of ribavirin on ANDV replication in the established lethal Syrian hamster model. We concluded that ribavirin treatment is beneficial for postexposure prophylaxis against HPS-causing hantaviruses (Safronetz 2011). (III) Ecology and Transmission Goal: Understanding the interaction of virus and reservoir species to prevent transmission into end host. We collected Mastomys natalensis in southern Mali and isolated an arenavirus. Genetic analysis confirmed a unique Lassa virus, Soromba R, and demonstrated for the first time the presence of Lassa in southern Mali (Safronetz 2010). We have developed a framework for animal and human filovirus surveillance in the Republic of Congo. In addition, facilities for basic research will be created for holding, quarantine and breeding of indigenous fruit bat species. This will allow pathogenicity and transmission studies in the potential reservoir species of filoviruses. We have already identified long-term sampling sites (Munster, in preparation). We have established an infection model for Sin nombre virus (hantavirus) to study the immune response to infection in the natural reservoir species, the deer mouse. We have found that the deer mouse mounts a strong anti-inflammatory CD4+ T cell response to infection, thereby limiting pathology and allowing virus persistence (Prescott, in preparation)